Selective Expression of KCNS3 Potassium Channel a-Subunit in Parvalbumin-Containing GABA Neurons in the Human Prefrontal Cortex Danko Georgiev1, Guillermo Gonza´lez-Burgos2, Mitsuru Kikuchi1,3, Yoshio Minabe1,3, David A. Lewis2,4, Takanori Hashimoto1,2* 1 Department of Psychiatry and Neurobiology, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan, 2 Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3 Research Center for Child Mental Development, Kanazawa University, Kanazawa, Ishikawa, Japan, 4 Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America Abstract The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV) or somatostatin (SST). Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K+ channel Kv9.3 modulatory a-subunit, LIM homeobox protein 6, K+ channel Kvb1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with 35S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in .90% of KCNS3 mRNA- expressing neurons. LHX6 mRNA was detected in almost all PV and .90% of SST neurons, while among all LHX6 mRNA- expressing neurons 50% expressed PV mRNA and .44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia. Citation: Georgiev D, Gonza´lez-Burgos G, Kikuchi M, Minabe Y, Lewis DA, et al. (2012) Selective Expression of KCNS3 Potassium Channel a-Subunit in Parvalbumin-Containing GABA Neurons in the Human Prefrontal Cortex. PLoS ONE 7(8): e43904. doi:10.1371/journal.pone.0043904 Editor: Takeo Yoshikawa, Rikagaku Kenkyu¯sho Brain Science Institute, Japan Received June 29, 2012; Accepted July 27, 2012; Published August 24, 2012 Copyright: ß 2012 Georgiev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Japan Society for the Promotion of Science (Postdoctoral Fellowship to DG and Grants-in-Aid 21390332, 21.09141 to TH) and National Institutes of Health (Grants MH043784, MH084053 to DAL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: DAL currently receives investigator-initiated research support from the National Institutes of Health, Commonwealth of Pennsylvania, Bristol-Myers Squibb, Curridium Ltd and Pfizer and in 2010–2012 served as a consultant in the areas of target identification and validation and new compound development to BioLine RX, Bristol-Myers Squibb, and Merck. TH served as a consultant for Ono Pharmaceutical Co., Ltd. in 2012. None of above funders was involved in the study design; collection, analysis, and interpretation of data; writing of the paper; or decision to submit for publication of this study. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: [email protected] Introduction and GAT1 appear to be prominent in PV- as well as SST- expressing GABA neuron subsets [6,9,10]. On the other hand, The core features of schizophrenia include disturbances in measures of the mRNA and protein for calretinin (CR), which is diverse cognitive functions that depend on the neural circuitry of expressed by a third subset of GABA neurons, were unaltered in the cerebral cortex [1]. In the cortex of subjects with schizophre- the cortex of subjects with schizophrenia [7,9,11,12,13,14]. nia, inhibitory neurotransmission mediated by c-aminobutyric Together, these findings indicate that cortical dysfunction in acid (GABA) appears to be altered [2], as indicated by lower levels schizophrenia selectively involves two separate subsets of GABA of the mRNAs encoding the 67 kilodalton isoform of glutamic acid neurons: PV and SST neurons. decarboxylase (GAD67) [3], the enzyme principally responsible for Understanding the molecular processes underlying the alter- GABA synthesis, and the GABA membrane transporter 1 (GAT1) ations in PV and SST neurons would be informed by identifying [4,5,6,7,8], which mediates the reuptake of synaptically released molecules that are selectively expressed in these neurons and that GABA. These alterations appear to involve specific subsets of contribute to their distinctive functions. The evaluation of such GABA neurons. For example, the mRNAs encoding parvalbumin molecules in schizophrenia might also reveal affected molecular (PV) and somatostatin (SST), each of which is expressed in a pathways in PV and/or SST neurons, which could be used for separate subset of cortical GABA neurons, are decreased in developing therapeutic strategies targeting selectively these neu- schizophrenia [7,9,10,11]. Moreover, the reductions in GAD67 PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43904 KCNS3 Expression in Cortical Parvalbumin Neurons rons. In order to identify such molecules, we first used published primer sets for the studied genes (Table S1). For LHX6 and gene expression data for mouse cortical neuron subsets and KCNAB1, which have several splice isoforms, amplified fragments selected 70 genes found to be either developmentally upregulated were located in the common region among all isoforms. or preferentially enriched in PV and/or SST neurons [15,16,17]. Nucleotide sequencing revealed 100% homologies for all amplified We then evaluated the expression patterns of these 70 genes in the fragments to the reported sequences in Genbank. These fragments online atlases of gene expression in the mouse or human cortex were subcloned into the plasmid pSTBlue-1 (Novagen, Madison, [18] and excluded genes that were detected in pyramidal-like WI). Antisense and sense probes were transcribed in vitro in the neurons with an apical dendrite, or that exhibited an apparently presence of 35S-CTP (PerkinElmer, Waltham, MA), using T7 or different laminar expression pattern from those of PV and/or SST SP6 RNA polymerase (Promega, Madison, WI). The templates mRNAs. We found that KCNS3, LHX6, KCNAB1 and PPP1R2 were then digested with RQ1 DNase (Promega), and riboprobes had cortical mRNA expression patterns similar to those of PV were purified by centrifugation through the RNeasy mini spin + and/or SST mRNAs. KCNS3 encodes voltage-gated K channel columns (Qiagen, Hilden, Germany). Hybridization was per- Kv9.3 modulatory a-subunit that coassembles with Kv2.1 formed as described previously [8,9,10]. For each gene, we a-subunits and leads to an enhanced conductance and modified processed at least 2 sections per subject. After fixation with 4% gating properties of the heteromeric channels [19,20,21]. LHX6 paraformaldehyde in PBS, the sections were acetylated with encodes LIM homeobox protein 6, a transcription factor suggested 0.25% acetic anhydrate in 0.1 M triethanolamine/0.9% NaCl for to be involved in the development of PV and SST neurons in the 10 min, and dehydrated through a graded ethanol series. The + mouse cortex [22,23]. KCNAB1 encodes K channel Kvb1 sections were then hybridized with 35S-labeled riboprobes accessory subunit that confers fast N-type inactivation to Kv1.1 (26107 dpm/ml) in hybridization buffer containing 50% form- channels [24]. PPP1R2 gene encodes protein phosphatase 1 (PP1) amide, 0.75 M NaCl, 20 mM 1,4-piperazine diethane sulfonic regulatory subunit 2, which inhibits PP1 and controls signal acid, pH 6.8, 10 mM EDTA, 10% dextran sulfate, 56Denhardt’s transduction and synaptic plasticity [25]. In this study, we solution (0.2 mg/ml Ficoll, 0.2 mg/ml polyvinylpyrrolidone, determined whether KCNS3, LHX6, KCNAB1 and PPP1R2 0.2 mg/ml BSA), 50 mM dithiothreitol, 0.2% SDS, and mRNAs are selectively expressed in PV and/or SST neurons in 100 mg/ml yeast tRNA at 56uC for 16 hr. The sections were the human prefrontal cortex (PFC). washed in a solution containing 0.3 M NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, and 50% formamide at 63uC, Materials and Methods treated with 20 mg/ml RNase A (Sigma-Aldrich, St Louis, MO) at 37 C, and washed in 0.16SSC (150 mM NaCl, 15 mM sodium Ethics Statement u citrate) at 66uC. Sections were then dehydrated through a graded All procedures were approved by the University of Pittsburgh ethanol series, air dried, and exposed to BioMax MR film (Kodak, Committee for Oversight of Research and Clinical Training Rochester, NY) for 5–10 days. After the exposure to film, sections Involving Decedents and Institutional Review Board for Biomed- were coated with NTB emulsion (Kodak) diluted 1:1 with distilled ical Research as well as by the Ethics Committee of Kanazawa water. To ensure the consistency of emulsion thickness across University Graduate School of Medical Science.